Protein-phospholipid Interactions Research Articles

Polar Localization Analysis of Anionic Phospholipids and Their Binding Proteins in Arabidopsis Pollen Tubes.

Pollen tubes are typical polarized growth cells whose elongation occurs only in tip regions and is highly dependent on precise and ordered exocytosis/endocytosis in the top regions of the tubes. Although anionic phospholipids have been proven to be involved in regulating vesicle trafficking and the proper localization and functions of proteins in pollen tubes, the underlying cellular and molecular mechanisms remain poorly understood. To further understand how anionic phospholipids are involved in vesicle trafficking and in the control of protein localization and functions, assay methods to analyze the polar localization of anionic phospholipids and their binding proteins, and identifying phospholipid-protein interactions, should be developed. Here, we describe detailed protocols for analyzing anionic phospholipid polar localization and colocalization with their binding proteins in Arabidopsis pollen tubes and examining phospholipid-protein interactions in vitro.

Study on the Stability Mechanism of Peanut OBs Extracted with the Aqueous Enzymatic Method.

In this study, the internal relationships among oil bodies (OBs), the protein-phospholipid interactions in aqueous phase, oil-water interface behavior, and the stability of reconstituted OBs were analyzed from the bulk phase, interface, and macro perspectives, and the stability mechanism of OBs was discussed. OB proteins and phospholipids were combined through hydrophobic and electrostatic interactions, resulting in the stretching of protein conformation. OB proteins and phospholipids act synergistically to increase interface pressure and the rate of increase in interface pressure with relatively stable elastic behavior, which is beneficial to the formation and stability of interfacial films. When OBs were reconstituted by an OB protein-phospholipid complex system, phospholipids bound to OB proteins through hydrophobic and electrostatic interactions. OB proteins and phospholipids uniformly covered the oil droplet surface of reconstituted OBs to form a stable interfacial film, which maintained the stability of OBs. The addition of phospholipids significantly reduced the particle size of OBs prepared by OB proteins in a dose-dependent manner, and particle size decreased with the increase in phospholipid content (p < 0.05). Phospholipids increased the net surface charge, enhanced electrostatic repulsion, and improved the physicochemical stability of reconstituted OBs. The stability mechanism elucidated in this study provides a theoretical basis for the demulsification of peanut OBs.

Selective expression of light-harvesting complexes alters phospholipid composition in the intracytoplasmic membrane and core complex of purple phototrophic bacteria

Phospholipid–protein interactions play important roles in regulating the function and morphology of photosynthetic membranes in purple phototrophic bacteria. Here, we characterize the phospholipid composition of intracytoplasmic membrane (ICM) from Rhodobacter (Rba.) sphaeroides that has been genetically altered to selectively express light-harvesting (LH) complexes. In the mutant strain (DP2) that lacks a peripheral light-harvesting (LH2) complex, the phospholipid composition was significantly different from that of the wild-type strain; strain DP2 showed a marked decrease in phosphatidylglycerol (PG) and large increases in cardiolipin (CL) and phosphatidylcholine (PC) indicating preferential interactions between the complexes and specific phospholipids. Substitution of the core light-harvesting (LH1) complex of Rba. sphaeroides strain DP2 with that from the purple sulfur bacterium Thermochromatium tepidum further altered the phospholipid composition, with substantial increases in PG and PE and decreases in CL and PC, indicating that the phospholipids incorporated into the ICM depend on the nature of the LH1 complex expressed. Purified LH1–reaction center core complexes (LH1–RC) from the selectively expressing strains also contained different phospholipid compositions than did core complexes from their corresponding wild-type strains, suggesting different patterns of phospholipid association between the selectively expressed LH1–RC complexes and those purified from native strains. Effects of carotenoids on the phospholipid composition were also investigated using carotenoid-suppressed cells and carotenoid-deficient species. The findings are discussed in relation to ICM morphology and specific LH complex–phospholipid interactions.

Using light scattering to assess how phospholipid-protein interactions affect complex I functionality in liposomes.

Complex I is an essential membrane protein in respiration, oxidising NADH and reducing ubiquinone to contribute to the proton-motive force that powers ATP synthesis. Liposomes provide an attractive platform to investigate complex I in a phospholipid membrane with the native hydrophobic ubiquinone substrate and proton transport across the membrane, but without convoluting contributions from other proteins present in the native mitochondrial inner membrane. Here, we use dynamic and electrophoretic light scattering techniques (DLS and ELS) to show how physical parameters, in particular the zeta potential (ζ-potential), correlate strongly with the biochemical functionality of complex I-containing proteoliposomes. We find that cardiolipin plays a crucial role in the reconstitution and functioning of complex I and that, as a highly charged lipid, it acts as a sensitive reporter on the biochemical competence of proteoliposomes in ELS measurements. We show that the change in ζ-potential between liposomes and proteoliposomes correlates linearly with protein retention and catalytic oxidoreduction activity of complex I. These correlations are dependent on the presence of cardiolipin, but are otherwise independent of the liposome lipid composition. Moreover, changes in the ζ-potential are sensitive to the proton motive force established upon proton pumping by complex I, thereby constituting a complementary technique to established biochemical assays. ELS measurements may thus serve as a more widely useful tool to investigate membrane proteins in lipid systems, especially those that contain charged lipids.

In vitro Assays to Evaluate Specificity and Affinity in Protein-phospholipid Interactions.

Protein-lipid interactions play important roles in many biological processes, including metabolism, signaling, and transport; however, computational and structural analyses often fail to predict such interactions, and determining which lipids participate in these interactions remains challenging. In vitro assays to assess the physical interaction between a protein of interest and a panel of phospholipids provide crucial information for predicting the functionality of these interactions in vivo. In this protocol, which we developed in the context of evaluating protein-lipid binding of the Arabidopsis thaliana florigen FLOWERING LOCUS T, we describe four independent in vitro experiments to determine the interaction of a protein with phospholipids: lipid-protein overlay assays, liposome binding assays, biotin-phospholipid pull-down assays, and fluorescence polarization assays. These complementary assays allow the researcher to test whether the protein of interest interacts with lipids in the test panel, identify the relevant lipids, and assess the strength of the interaction.

Characterization of Protein-Phospholipid/Membrane Interactions Using a "Membrane-on-a-Chip" Microfluidic System.

It is now clear that organelles of a mammalian cell can be distinguished by phospholipid profiles, both as ratios of common phospholipids and by the absence or presence of certain phospholipids. Organelle-specific phospholipids can be used to provide a specific shape and fluidity to the membrane and/or used to recruit and/or traffic proteins to the appropriate subcellular location and to restrict protein function to this location. Studying the interactions of proteins with specific phospholipids using soluble derivatives in isolation does not always provide useful information because the context in which the headgroups are presented almost always matters. Our laboratory has shown this circumstance to be the case for a viral protein binding to phosphoinositides in solution and in membranes. The system we have developed to study protein-phospholipid interactions in the context of a membrane benefits from the creation of tailored membranes in a channel of a microfluidic device, with a fluorescent lipid in the membrane serving as an indirect reporter of protein binding. This system is amenable to the study of myriad interactions occurring at a membrane surface as long as a net change in surface charge occurs in response to the binding event of interest.

Phosphatidylinositol 4,5-bisphosphate directly interacts with the β and γ subunits of the sodium channel ENaC

The plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of diverse ion channels to include the epithelial Na+ channel ENaC. Whether PIP2 regulation of ENaC is due to a direct phospholipid-protein interaction, remains obscure. To date, possible interaction of PIP2 with ENaC primarily has been tested indirectly through assays of channel function. A fragment-based biochemical analysis approach is used here to directly quantify possible PIP2-ENaC interactions. We find using the CIBN-CRY2 optogenetic dimerization system that the phosphoryl group positioned at carbon 5 of PIP2 is necessary for interaction with ENaC. Previous studies have implicated conserved basic residues in the cytosolic portions of β- and γ-ENaC subunits as being important for PIP2-ENaC interactions. To test this, we used synthetic peptides of these regions of β- and γ-ENaC. Steady-state intrinsic fluorescence spectroscopy demonstrated that phosphoinositides change the local conformation of the N terminus of β-ENaC, and two sites of γ-ENaC adjacent to the plasma membrane, suggesting direct interactions of PIP2 with these three regions. Microscale thermophoresis elaborated PIP2 interactions with the N termini of β- (Kd ∼5.2 μm) and γ-ENaC (Kd ∼13 μm). A weaker interaction site within the carboxyl terminus of γ-ENaC (Kd ∼800 μm) was also observed. These results support that PIP2 regulates ENaC activity by directly interacting with at least three distinct regions within the cytoplasmic domains of the channel that contain conserved basic residues. These interactions are probably electrostatic in nature, and are likely to bear a key structural role in support of channel activity.

Interactions of Leptospiral PI-PLC with the membrane phosphoinositides: an insight of the proteinphospholipid association in the pathogenesis

Protein-phospholipid interactions encompass the basis of many biological processes, including host-pathogen interaction and disease progression. This study reports the interactions of Leptospiral PI-PLC (LA_1375) with PIP2 and PIP3. Molecular docking analysis was carried out to unveil the interactions of the protein with the plasma membrane phosphoinositides. The binding affinities of the interactions, most favourable protein-phospholipid binding poses, and the binding site residues were revealed. The binding energies were found to be -14.04 and -11.64 kcal/mol respectively for PIP2 and PIP3 interaction. Also, the analysis explored the high-affinity binding of the protein with the substrate PIP2. PIP2 is the precursor of intracellular second messengers DAG and IP3; responsible for the activation of pro-inflammatory mediators and other signalling events. Moreover, both PIP2 and PIP3 have been implicated in various cellular processes through the activation of lipid signalling cascades. Hence, further in-vitro confirmation of the annotated interactions may open up new dimensions in the pathogenesis of Leptospirosis.

Interactions of Leptospiral PI-PLC with the membrane phosphoinositides: an insight of the protein-phospholipid association in the pathogenesis

Protein-phospholipid interactions encompass the basis of many biological processes, including host-pathogen interaction and disease progression. This study reports the interactions of Leptospiral PI-PLC (LA_1375) with PIP2 and PIP3. Molecular docking analysis was carried out to unveil the interactions of the protein with the plasma membrane phosphoinositides. The binding affinities of the interactions, most favourable protein-phospholipid binding poses, and the binding site residues were revealed. The binding energies were found to be -14.04 and -11.64 kcal/mol respectively for PIP2 and PIP3 interaction. Also, the analysis explored the high-affinity binding of the protein with the substrate PIP2. PIP2 is the precursor of intracellular second messengers DAG and IP3; responsible for the activation of pro-inflammatory mediators and other signalling events. Moreover, both PIP2 and PIP3 have been implicated in various cellular processes through the activation of lipid signalling cascades. Hence, further in-vitro confirmation of the annotated interactions may open up new dimensions in the pathogenesis of Leptospirosis.

Transient Gene Expression as a Tool to Monitor and Manipulate the Levels of Acidic Phospholipids in Plant Cells.

Anionic phospholipids represent only minor fraction of cell membranes lipids but they are critically important for many membrane-related processes, including membrane identity, charge, shape, the generation of second messengers, and the recruitment of peripheral proteins. The main anionic phospholipids of the plasma membrane are phosphoinositides phosphatidylinositol 4-phosphate (PI4P), phosphatidylinositol 4,5-bisphosphate (PI4,5P2), phosphatidylserine (PS), and phosphatidic acid (PA). Recent insights in the understanding of the nature of protein-phospholipid interactions enabled the design of genetically encoded fluorescent molecular probes that can interact with various phospholipids in a specific manner allowing their imaging in live cells. Here, we describe the use of transiently transformed plant cells to study phospholipid-dependent membrane recruitment.

Membrane remodeling in clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is an essential cellular mechanism by which all eukaryotic cells regulate their plasma membrane composition to control processes ranging from cell signaling to adhesion, migration and morphogenesis. The formation of endocytic vesicles and tubules involves extensive protein-mediated remodeling of the plasma membrane that is organized in space and time by protein-protein and protein-phospholipid interactions. Recent studies combining high-resolution imaging with genetic manipulations of the endocytic machinery and with theoretical approaches have led to novel multifaceted phenomenological data of the temporal and spatial organization of the endocytic reaction. This gave rise to various - often conflicting - models as to how endocytic proteins and their association with lipids regulate the endocytic protein choreography to reshape the plasma membrane. In this Review, we discuss these findings in light of the hypothesis that endocytic membrane remodeling may be determined by an interplay between protein-protein interactions, the ability of proteins to generate and sense membrane curvature, and the ability of lipids to stabilize and reinforce the generated membrane shape through adopting their lateral distribution to the local membrane curvature.

Plant Phospholipid Diversity: Emerging Functions in Metabolism and Protein-Lipid Interactions.

Phospholipids are essential components of biological membranes and signal transduction cascades in plants. In recent years, plant phospholipid research was greatly advanced by the characterization of numerous mutants affected in phospholipid biosynthesis and the discovery of a number of functionally important phospholipid-binding proteins. It is now accepted that most phospholipids to some extent have regulatory functions, including those that serve as constituents of biological membranes. Phospholipids are more than an inert end product of lipid biosynthesis. This review article summarizes recent advances on phospholipid biosynthesis with a particular focus on polar head group synthesis, followed by a short overview on protein-phospholipid interactions as an emerging regulatory mechanism of phospholipid function in arabidopsis (Arabidopsis thaliana).

Structural analysis of the interaction between Jaburetox, an intrinsically disordered protein, and membrane models

Jack bean urease is entomotoxic to insects with cathepsin-like digestive enzymes, and its toxicity is mainly caused by a polypeptide called Jaburetox (Jbtx), released by cathepsin-dependent hydrolysis of the enzyme. Jbtx is intrinsically disordered in aqueous solution, as shown by CD and NMR. Jbtx is able to alter the permeability of membranes, hinting to a role of Jbtx-membrane interaction as the basis for its toxicity. The present study addresses the structural aspects of this interaction by investigating the behaviour of Jbtx when in contact with membrane models, using nuclear magnetic resonance and circular dichroism spectroscopies in the absence or presence of micelles, large unilamellar vesicles, and bicelles. Fluorescence microscopy was also used to detect protein-insect membrane interaction. Significant differences were observed depending on the type of membrane model used. The interaction with negatively charged SDS micelles increases the secondary and tertiary structure content of the polypeptide, while, in the case of large unilamellar vesicles and bicelles, conformational changes were observed at the terminal regions, with no significant acquisition of secondary structure motifs. These results were interpreted as suggesting that the Jbtx-lipids interaction anchors the polypeptide to the cellular membrane through the terminal portions of the polypeptide and that, following this interaction, Jbtx undergoes conformational changes to achieve a more ordered structure that could facilitate its interaction with membrane-bound proteins. Consistently with this hypothesis, the presence of these membrane models decreases the ability of Jbtx to bind cellular membranes of insect nerve cord. The collected evidence from these studies implies that the biological activity of Jbtx is due to protein-phospholipid interactions.

Protein-phospholipid interplay revealed with crystals of a calcium pump.

The lipid bilayer has so far eluded visualization by conventional crystallographic methods, severely limiting our understanding of phospholipid- and protein-phospholipid interactions. Here we describe electron density maps for crystals of Ca2+-ATPase in four different states obtained by X-ray solvent contrast modulation. These maps resolve the entire first layer of phospholipids surrounding the transmembrane helices, although less than half of them are hydrogen-bonded to protein residues. Phospholipids follow the movements of associated residues, causing local distortions and changes in thickness of the bilayer. Unexpectedly, the entire protein tilts during the reaction cycle, governed primarily by a belt of Trp residues, to minimize energy costs accompanying the large perpendicular movements of the transmembrane helices. A class of Arg residues extend their side chains through the cytoplasm to exploit phospholipids as anchors for conformational switching. Thus, phospholipid-Arg/Lys and phospholipid-Trp interactions have distinct functional roles in the dynamics of ion pumps and, presumably, membrane proteins in general.

Global Mapping of Protein–Lipid Interactions by Using Modified Choline‐Containing Phospholipids Metabolically Synthesized in Live Cells

AbstractThe protein–lipid interaction is an essential metabolic process that mediates cellular signaling and functions. Existing strategies for large‐scale mapping studies of the protein–lipid interaction fall short in their incompatibility with metabolic incorporation or inability to remove unwanted interferences from lipidated proteins. By incorporating an alkyne‐containing choline head group and a diazirine‐modified fatty acid simultaneously into choline‐containing phospholipids synthesized from live mammalian cells, protein–phospholipid interactions have been successfully imaged in live cells. Subsequent in situ profiling of the modified Cho phospholipid‐crosslinked proteins followed by quantitative proteomics allowed identification of several hundred putative phospholipid‐interacting proteins, some of which were further validated.

Global Mapping of Protein-Lipid Interactions by Using Modified Choline-Containing Phospholipids Metabolically Synthesized in Live Cells.

The protein-lipid interaction is an essential metabolic process that mediates cellular signaling and functions. Existing strategies for large-scale mapping studies of the protein-lipid interaction fall short in their incompatibility with metabolic incorporation or inability to remove unwanted interferences from lipidated proteins. By incorporating an alkyne-containing choline head group and a diazirine-modified fatty acid simultaneously into choline-containing phospholipids synthesized from live mammalian cells, protein-phospholipid interactions have been successfully imaged in live cells. Subsequent in situ profiling of the modified Cho phospholipid-crosslinked proteins followed by quantitative proteomics allowed identification of several hundred putative phospholipid-interacting proteins, some of which were further validated.

Membrane Perturbation of ADP-insensitive Phosphoenzyme of Ca2+-ATPase Modifies Gathering of Transmembrane Helix M2 with Cytoplasmic Domains and Luminal Gating

Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase involves ATP-dependent phosphorylation of a catalytic aspartic acid residue. The key process, luminal Ca2+ release occurs upon phosphoenzyme isomerization, abbreviated as E1PCa2 (reactive to ADP regenerating ATP and with two occluded Ca2+ at transport sites) → E2P (insensitive to ADP and after Ca2+ release). The isomerization involves gathering of cytoplasmic actuator and phosphorylation domains with second transmembrane helix (M2), and is epitomized by protection of a Leu119-proteinase K (prtK) cleavage site on M2. Ca2+ binding to the luminal transport sites of E2P, producing E2PCa2 before Ca2+-release exposes the prtK-site. Here we explore E2P structure to further elucidate luminal gating mechanism and effect of membrane perturbation. We find that ground state E2P becomes cleavable at Leu119 in a non-solubilizing concentration of detergent C12E8 at pH 7.4, indicating a shift towards a more E2PCa2-like state. Cleavage is accelerated by Mg2+ binding to luminal transport sites and blocked by their protonation at pH 6.0. Results indicate that possible disruption of phospholipid-protein interactions strongly favors an E2P species with looser head domain interactions at M2 and responsive to specific ligand binding at the transport sites, likely an early flexible intermediate in the development towards ground state E2P.

SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment.

Endocytosis defines the entry of molecules or macromolecules through the plasma membrane as well as membrane trafficking in the cell. It depends on a large number of proteins that undergo protein-protein and protein-phospholipid interactions. EH Domain containing (EHDs) proteins formulate a family, whose members participate in different stages of endocytosis. Of the four mammalian EHDs (EHD1-EHD4) EHD1 and EHD3 control traffic to the endocytic recycling compartment (ERC) and from the ERC to the plasma membrane, while EHD2 modulates internalization. Recently, we have shown that EHD2 undergoes SUMOylation, which facilitates its exit from the nucleus, where it serves as a co-repressor. In the present study, we tested whether EHD3 undergoes SUMOylation and what is its role in endocytic recycling. We show, both in-vitro and in cell culture, that EHD3 undergoes SUMOylation. Localization of EHD3 to the tubular structures of the ERC depends on its SUMOylation on lysines 315 and 511. Absence of SUMOylation of EHD3 has no effect on its dimerization, an important factor in membrane localization of EHD3, but has a dominant negative effect on its appearance in tubular ERC structures. Non-SUMOylated EHD3 delays transferrin recycling from the ERC to the cell surface. Our findings indicate that SUMOylation of EHD3 is involved in tubulation of the ERC membranes, which is important for efficient recycling.

Dynamic phospholipid interaction of β2e subunit regulates the gating of voltage-gated Ca2+ channels.

High voltage-activated Ca2+ (Ca(V)) channels are protein complexes containing pore-forming α1 and auxiliary β and α2δ subunits. The subcellular localization and membrane interactions of the β subunits play a crucial role in regulating Ca(V) channel inactivation and its lipid sensitivity. Here, we investigated the effects of membrane phosphoinositide (PI) turnover on Ca(V)2.2 channel function. The β2 isoform β2e associates with the membrane through electrostatic and hydrophobic interactions. Using chimeric β subunits and liposome-binding assays, we determined that interaction between the N-terminal 23 amino acids of β2e and anionic phospholipids was sufficient for β2e membrane targeting. Binding of the β2e subunit N terminus to liposomes was significantly increased by inclusion of 1% phosphatidylinositol 4,5-bisphosphate (PIP2) in the liposomes, suggesting that, in addition to phosphatidylserine, PIs are responsible for β2e targeting to the plasma membrane. Membrane binding of the β2e subunit slowed Ca(V)2.2 current inactivation. When membrane phosphatidylinositol 4-phosphate and PIP2 were depleted by rapamycin-induced translocation of pseudojanin to the membrane, however, channel opening was decreased and fast inactivation of Ca(V)2.2(β2e) currents was enhanced. Activation of the M1 muscarinic receptor elicited transient and reversible translocation of β2e subunits from membrane to cytosol, but not that of β2a or β3, resulting in fast inactivation of Ca(V)2.2 channels with β2e. These results suggest that membrane targeting of the β2e subunit, which is mediated by nonspecific electrostatic insertion, is dynamically regulated by receptor stimulation, and that the reversible association of β2e with membrane PIs results in functional changes in Ca(V) channel gating. The phospholipid-protein interaction observed here provides structural insight into mechanisms of membrane-protein association and the role of phospholipids in ion channel regulation.

The Conformational Dynamics of a Secondary Multidrug Transporter are Modulated by the Lipid Bilayer Composition

LmrP, a Major Facilitator Superfamily (MFS) multidrug transporter from Lactococcus lactis, acts as a secondary antiporter, catalysing the extrusion of a spectrum of hydrophobic drugs by dissipating a proton gradient. It has evolved to bind and export structurally diverse cytotoxic substances from within the membrane, in contrast to the majority of MFS transporters specialized in the transport of a single soluble substrate, captured from the aqueous medium. Its transport mechanism could thus differ from the alternating access model of the lactose permease, the actual paradigm for all MFS transporters. Recently, we have shown that the protonation of conserved acidic residues is the driving force of the conformational transition, and that substrate binding initiates the cycle by catalysing proton passage (Masureel, Martens et al. 2014). While this analysis was performed on detergent solubilized protein, we now aim to investigate the role of the lipid bilayer in the conformational cycle. For this purpose, we performed Double Electron Electron Resonance (DEER) distance measurements on a library of labeled double-cysteine mutants monitoring both sides of the transporter. Spin labeled mutants were reconstituted in nanodiscs and the distance changes monitored upon changes in the pH or addition of substrate(s). We found that the pH-dependent equilibrium between the inside-open and outside-open conformations is strongly shifted in nanodiscs compared to detergent, suggesting that the pKa of the acidic residues is modified by the lipids. By combining specific lipidic compositions and mutagenesis, we are investigating whether such change in the dynamics are the results of direct phospholipid-protein interaction. Specific structural effects of given lipids are observed: small amounts of cardiolipin allow the conformational transition to occur at physiological pH. These findings could shed light on the mechanism of MFS multidrug transporters in general.